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Philos Trans R Soc Lond B Biol Sci. 2018 Jun 5;373(1748). pii: 20170083. doi: 10.1098/rstb.2017.0083.

New chemical tools for probing activity and inhibition of the NAD+-dependent lysine deacylase sirtuin 2.

Author information

1
Institute of Pharmaceutical Sciences, University of Freiburg, Albertstraße 19, 79104 Freiburg im Breisgau, Germany.
2
Department of Chemistry, Chemistry Research Laboratory, University of Oxford, Mansfield Road, Oxford OX1 3TA, UK.
3
Institute of Enzymology, Research Centre for Natural Sciences, Hungarian Academy of Sciences, Magyar Tudósok körútja 2, 1117, Budapest, Hungary.
4
Department of Chromatin Regulation, Max-Planck-Institute of Immunobiology and Epigenetics, Stübeweg 51, 79108 Freiburg im Breisgau, Germany.
5
Institute of Pharmacy, Martin-Luther-University Halle-Wittenberg, Wolfgang-Langenbeck-Straße 4, 06120 Halle (Saale), Germany.
6
Institute of Pharmaceutical Sciences, University of Freiburg, Albertstraße 19, 79104 Freiburg im Breisgau, Germany manfred.jung@pharmazie.uni-freiburg.de.
7
Freiburg Institute of Advanced Studies (FRIAS), University of Freiburg, Albertstraße 19, 79104 Freiburg im Breisgau, Germany.

Abstract

Sirtuins are NAD+-dependent protein deacylases capable of cleaving off acetyl as well as other acyl groups from the ɛ-amino group of lysines in histones and other substrate proteins. They have been reported as promising drug targets, and thus modulators of their activity are needed as molecular tools to uncover their biological function and as potential therapeutics. Here, we present new assay formats that complement existing assays for sirtuin biochemistry and cellular target engagement. Firstly, we report the development of a homogeneous fluorescence-based activity assay using unlabelled acylated peptides. Upon deacylation, the free lysine residue reacts with fluorescamine to form a fluorophore. Secondly, using click chemistry with a TAMRA-azide on a propargylated sirtuin inhibitor, we prepared the first fluorescently labelled small-molecule inhibitor of Sirt2. This is used in a binding assay, which is based on fluorescence polarization. We used it successfully to map potential inhibitor-binding sites and also to show cellular Sirt2 engagement. By means of these new assays, we were able to identify and characterize novel Sirt2 inhibitors out of a focused library screen. The binding of the identified Sirt2 inhibitors was rationalized by molecular docking studies. These new chemical tools thus can enhance further sirtuin research.This article is part of a discussion meeting issue 'Frontiers in epigenetic chemical biology'.

KEYWORDS:

NAD+; Sirt2; assays; deacetylases; epigenetics; sirtuins

PMID:
29685963
PMCID:
PMC5915722
DOI:
10.1098/rstb.2017.0083
[Indexed for MEDLINE]
Free PMC Article

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